Rabbit anti akt antibody

Cells were lysed using SDS-PAGE loading buffer without reducing agents. Proteins were separated by SDS-PAGE and transferred to a nitrocellulose membrane. Antibodies were applied according to their manufacturers’ protocols. Blots were developed with SuperSignal West Pico Substrate (Thermo Fisher Scientific, Montreal, QC). Anti-phospho-ERK1/2 MAPK (T²⁰²/Y²⁰⁴) mouse antibody (1:1000), anti-ERK1/2 MAPK mouse antibody (1:1000), anti-phospho-Akt (S⁴⁷³) rabbit antibody (1:1000), anti-Akt rabbit antibody (1:1000), anti-phospho-c-Jun (S⁶³) rabbit antibody (1:1000), anti-c-Jun rabbit antibody (1:1000), anti-phospho-p65 (S⁵³⁶) rabbit antibody (1:1000), anti-p65 rabbit antibody (1:1000), anti-phospho-HSP27 (S⁸²) rabbit antibody (1:1000) and anti-HSP27 rabbit antibody (1:1000) were obtained from Cell Signaling Technology (Beverly, MA), Anti-E-selectin mouse monoclonal antibody (1:1000) was obtained from R&D Systems (Minneapolis, MN). Anti-METTL3 rabbit antibody (1:2000) was obtained from Abcam (Cambridge, UK). Anti-GAPDH mouse antibody (1:10000) was obtained from Novus Biologicals (Oakville, ON). Anti-mouse/rabbit-IgG-horseradish-peroxidase (HRP) goat antibodies (1:5000) were obtained from The Jackson Laboratory (Bar Harbor, ME).

The protein levels of AKT, phosphorylated AKT, BCL2, BAK and BAX were determined by Western immunoblotting analysis as previously described [34 (link)]. In brief, proteins isolated from cells (50 μg) were loaded and separated by electrophoresis on a 10% SDS polyacrylamide gel and then transferred to a nitrocellulose membrane. The membrane was probed with either rabbit anti-Akt antibody (Cell Signaling Technology), rabbit anti-phosphorylated AKT antibody (Cell Signaling Technology), rabbit anti-BAK antibody (Cell Signaling Technology), rabbit anti-BAX antibody (Cell Signaling Technology), or mouse anti-BCL2 antibody (Neomarkers). All antibodies were diluted at 1:1000. HRP-conjugated anti-rabbit or anti-mouse IgG antibody (Jackson ImmunoResearch) was used as the secondary antibody. The corresponding protein bands were visualized using enhanced chemiluminescence reagents. The same membranes were re-probed with rabbit anti-β-actin monoclonal antibody (Abcam) to confirm equal loading of proteins for each sample.

BMDMs were stimulated with 100 ng/ml LPS for 1 h at 37 °C. In some cases, LY-294002 (3 μM) or wortmannin (300 nM) was applied 30 min before the LPS stimulation. BMDMs were homogenized in lysis buffer (pH 7.4) as described previously^{47 (link)}. The samples (20 μl/lane) were then subjected to electrophoresis on 10% SDS-polyacrylamide gels and transferred electrophoretically to PVDF membranes (Millipore, Billerica, MA). The membranes were incubated with rabbit anti-pAkt (Ser473) antibody, rabbit anti-Akt antibody, rabbit anti-p-GSK-3β (Ser9) antibody or rabbit anti-PI3 Kinase p85 antibody (Cell Signaling Technology, Denvers, MA) and treated with horseradish peroxidase-conjugated goat anti-rabbit IgG (Cell Signaling Technology). Immune complexes were visualized using an enhanced chemiluminescence detection system (GE Healthcare Japan, Tokyo, Japan) and photographed (ImageQuant LAS4000; GE Healthcare Japan). Akt and pAkt protein expression levels were determined densitometrically with ImageJ (NIH, Bethesda, MD).

Co-IPs were performed according to a standard protocol using a Pierce co-IP Kit (ThermoFisher Scientific). Briefly, differently pre-treated cells were harvested in ice-cold IP Lysis/Wash Buffer, before centrifugation at 13 000 × g for 10 min to pellet the cell debris. Then, the IP was performed by the addition of antibodies to cell lysates: rabbit anti-AKT antibody (Cell Signaling Technology) to IP the SRC-3 protein, and rabbit anti-SRC-3 antibody (Cell Signaling Technology) to IP the AKT protein. All co-IP steps were performed at 4 °C unless otherwise indicated. Subsequently, protein A/G beads (Thermo Fisher Scientific) were added for an additional 2 h. The immunoprecipitated proteins were washed five times with IP Lysis/Wash Buffer. Finally, proteins were resolved by SDS/PAGE and immunoblotted with antibodies as indicated.

Cells were harvested in ice-cold PBS, pelleted at 800 g (for HepG2 and AML-12 cells) or 50 g (for primary hepatocytes), and lysed for 1 h on ice in 1% Triton X-100, 0.2% SDS, 1 mM EDTA, 3.3 mM EGTA 1 mM Na₃VO₄, 15 mM NaF, 2 mM Na₄P₂O₇, 0.8 mM β-glycerophosphate, and 10 mM Tris-HCl, pH 7.4. Lysates were centrifuged at 15,000g for 10 min at 4°C and the clear supernatant was used as cell extracts. The protein concentration was measured by Lowry assay. Proteins (50 μg/lane) were resolved by 7.5% or 10% SDS-PAGE and transferred to Immobilon-P PVDF membrane by semidry blotting. The membranes were blocked in Tris-buffered saline (TBS, pH 7.4) containing 5% BSA and 0.05% Tween-20. pAkt and total Akt were detected by incubating the membrane with rabbit anti-Phospho-Akt (Ser473) or rabbit anti-Akt antibody (Cell Signaling Technology, Inc, Danvers, MA) at a dilution of 1:1000 in 1% BSA and 0.05% Tween-20 at 4°C overnight followed by anti-rabbit IgG-alkaline phosphatase-conjugated antibody (Sigma-Aldrich, Catalog # A3812) at a dilution of 1:10,000 at room temperature for 1.0 h. Protein antibody interactions were visualized using the ECF kit (Cytiva, Catalog # RPN5785) and ChemiDoc Imager (Bio-Rad Laboratories, Hercules, CA). Pierce Restore Western blotting stripping buffer (Thermo Fisher Scientific, Waltham, MA) was used when needed.